Kinetic mechanisms of rat polymerase beta-ssDNA interactions. Quantitative fluorescence stopped-flow analysis of the formation of the (Pol beta)(16) and (Pol beta)(5) ssDNA binding mode

摘要

Kinetics of rat polymerase beta (pol beta) binding to the single-stranded DNA (ssDNA) in the (pol beta)(16) and (pol beta)(5) binding modes has been examined, using the fluorescence stopped-flow technique. Binding of the enzyme to the ssDNA containing fluorescein is characterized by a strong increase of the DNA fluorescence, which provides an excellent signal to quantitatively study the complex mechanism of the ssDNA recognition process. The experiments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol beta)(16) binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol beta, and with a 10-mer which binds the enzyme exclusively in the (pol beta)(5) binding mode where only the 8 kDa domain of the enzyme is engaged in interactions with the DNA. The data indicate that the formation of the (pol beta)(16) binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations: